1. Field PA1 a. The PPE should be capable of luminescent in solutions of at least 80 percent water. PA1 b. The molecular efficiency of the luminescent reaction should be greater than or equal to 0.005. Molecular efficiency is defined as the number of photons emitted divided by the number of molecules excited. A more complete discussion on molecular efficiency can be found in Analytical Chemistry, Vol. 46, No. 2, p. 188A, February (1974), by Seitz & Neary entitled: "Chemiluminescence and Bioluminescence."
This invention is directed to a composition and method for chemical analysis and particularly to a tracer composition comprising a nonradioactive photon emitter coupled to a ligand and its corresponding selective ligator or binding body.
2. State of the Art
The use of radioactive tracers with photon emitting materials for detecting and/or measuring organic materials is known as evidenced by the above referred to co-pending patent application. In that patent application an insolubilized or solid phosphor and binding body, such as an antibody, are associated by chemical or physical means to provide a solid scintillating immunoadsorbent composition. This composition is capable of selectively binding or retaining a radioactive labelled substance to a phosphor or photon emitting substance. The luminescence emitted by the phosphor is measured by a scintillation counter and is directly proportional to the radioactive energy released by the labeled antigen bound to the antibody that is coupled to the phosphor. A convenient means for combining a solid phosphor and/or binding agent is to covalently couple the binding body to the surface of the phosphor which is in bead form. Also, these beads could have ferrous metal or magnetic material included in their composition for magnetic manipulation during an assay.
A new radioactove tracer composition could be made by combining scintillating phosphors with a suitable polymeric substance and forming insoluble particles, beads or filaments a few microns in diameter. Included in their composition would be one or more insolubilized, radioactive isotopes which would constantly activate the phosphors within. Also, these particles could have ferrous metal or magnetic material included in their composition for magnetic manipulation during an assay.
The surface of these micro-particles could be derivatized or modified chemically so that specific antibodies (or antigens) could be covalently coupled to their surface. The resulting composition would be constantly scintillating particles with the specific binding properties of the antibody (or antigen) coupled to their surfaces. These particles could be used as a suspension of tracer particles in various competitive or noncompetitive radioimmunoassay methods.
Also, isotopes with different energies, such as .sup.3 H, .sup.35 S and .sup.14 C or different phosphors that emit distinguishable wavelengths could be used to produce tracer particles of different "colors". By coupling one type of antibody (or antigen) with particles of one color, they could be distinguished from particles of another color and antibody in the same suspension. In this way one or more substances could be assayed simultaneously in the same sample using a scintillation spectrophotometer.
Although the above patent application was directed primarily to the use of labeled antigens capable of transmitting radioactive energy to energize a photon emitting substance and thereby produce a measurable amount of luminescence; luminescence may also be produced by other means. For example, luminescence may be produced by chemical or biological means and does not require the use of radioactive energy. The resulting chemiluminescence or bioluminescence is capable of being measured by conventional measuring means such as a photometer, or by using photographic film.
In addition to radioactive tracers or radioactive induced luminescence, the literature discloses the use of fluorometric tracers for quantitatively and qualitatively measuring a chemical reaction.
A "tracer" is defined broadly as a material that is directly or indirectly detectable during or upon completion of a chemical or biological reaction. This is generally achieved by labelling an atom or molecule of one of the reactants used in the reaction. In a ligand binding reaction, a tracer is produced by combining a detectable substance with a ligand or ligator without adversely affecting its chemical or biological properties. Two major areas where ligand binding is employed are in chemical and clinical assays such as in immunoassays and in chemical separation assays as in chromatography.
Radioactive tracers have been used in "competitive" and "noncompetitive" assays wherein tracer-ligands are utilized in binding reactions and subsequently measured. The use of radioactive tracers was demonstrated in the above referred to co-pending patent application.
Fluorometric tracers have been used primarily in the form of fluorescent dyes wherein the dyes are absorbed or otherwise attached to a reactant. An incident light is then applied to activate the fluorescent product.
Although the use of radioactive and fluorometric materials as tracers are known, very little, if anything, can be found whereby a luminescent material is used as a tracer. One reason is that in the coupling of a luminescent material to a ligand, a coupling agent is usually needed. However, with certain radioactive and fluorescent tracers this combination can be achieved with relative ease.
With radioactive or fluorescent techniques, certain inherent disadvantages exist. For example, in using radioactive tracers, one disadvantage is that the isotopes used therein are inherently unstable since they are detected only when they disintegrate and therefore will make the tracer itself unstable. The highly active emitters are more desirable since they do not require highly sophisticated equipment to be detected as do the low energy emitters. However, the highly active emitters usually have shorter lifetimes, are more of a radiation hazard to workers and usually require stricter licensing for their use.
The disadvantage associated with fluorometric tracers is that the fluorometric tracers require an incident light source to activate the material. This contributes background interference which necessarily increases instrument complexity and expense in controlling this problem. In addition, certain filters or refraction gratings and quartz cells are normally required in order to measure the degree of energy being emitted.
Enzyme immunoassays are also well known wherein common enzymes such as oxidases, dehydrogenases and reductases have been used as labels for ligands. However, biological fluids such as human serum frequently contain enzymes with similar activity to the enzyme tracer being used. These serum enzymes can frequently be a source of troublesome background activity and require additional sample processing for their removal.